title: Amplification and sequencing of the hepatitis C virus NS3/4A protease and the NS5B polymerase regions for genotypic resistance detection of clinical isolates of subtypes 1a and 1b.
abstract: Genotypic testing based on subtype-specific amplification and population Sanger sequencing for two nonstructural (NS) protein-coding regions, the NS3/4A protease and the NS5B polymerase, of the hepatitis C virus (HCV) genome is described here. The protocols include the molecular steps for RNA extraction, one-step RT-PCR followed by inner PCR and population Sanger sequencing, to obtain the sequence information of the target regions from the clinical isolates of HCV subtypes 1a and 1b, which can be used to detect any sequence change in the viral genome as for example caused by the development of drug resistance in these two common viral targets.
authors.affiliation: Janssen Diagnostics BVBA, Beerse, Belgium.
authors.forename: Diana
authors.initials: D
authors.lastname: Koletzki
authors.forename: Theresa
authors.initials: T
authors.lastname: Pattery
authors.forename: Bart
authors.initials: B
authors.lastname: Fevery
authors.forename: Leen
authors.initials: L
authors.lastname: Vanhooren
authors.forename: Lieven J
authors.initials: LJ
authors.lastname: Stuyver
mesh_terms: D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase